Vaccines for immunization against Helicobacter

ABSTRACT

The invention relates to the immunisation of pigs against Candidatus  Helicobacter suis  using antigens of species related to Candidatus  Helicobacter suis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.provisional patent application No. 60/691,394, filed Jun. 16, 2005, andU.S. provisional patent application No. 60/695,995, filed Jul. 1, 2005,the disclosures of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to tools and methods for the immunisationof animals against infection by Helicobacter species.

BACKGROUND OF THE INVENTION

Helicobacter (H.) pylori infections in humans are a major cause ofgastric and duodenal ulceration as well as gastric cancer. Tripletherapies with proton pump inhibitors and clarithromycin and amoxicillinare recommended as first line treatment. These standard therapiesincreasingly face problems with antibiotic resistance and recurrence ofinfection, especially in areas where H. pylori is endemic. Variousstudies in animal models have shown the feasibility of both prophylacticand therapeutic vaccination against H. pylori (Del Guidice (2001) Annu.Rev. Immunol. 19, 523-563; Sanchez et al. (2001) FEMS Immunol MedMicrobiol. 30, 157-165). H. pylori proteins expressed in infected miceand hence exposed to the mouse immune system, appear similar to thosedetected in human infections, suggesting that the mouse model issuitable for the preclinical screening of antigen candidates (Bumann etal. (2002) Inf. Imm. 70, 6494-6498). Immunizations with recombinanturease was found to induce local and serum immune responses in mice andprotect against Helicobacter pylori infection (Kleanthous et al. (1998)Inf. Imm. 66, 2879-2886). H. pylori is not the only bacterial pathogencapable of colonizing the human gastric mucosa. “H. heilmannii” indeedhas been found in approximately 0.96% of gastric biopsies. This organismis strongly associated with gastritis, but also with peptic ulceration,gastric adenocarcinoma and mucosa associated lymphoid tissue (MALT)lymphoma.

Some studies revealed sufficient antigenic cross-reactivity between H.felis and H. pylori to generate protection to H. felis challengefollowing immunization with a H. pylori sonicated antigen solution (Lee& Chen (1994) Inf. Imm. 62, 3594-3597; Michettti et al. (1994)Gastroenterology 107, 1002-1011). One study shows that H. heilmanniiinfection can be prevented by vaccination both with H. heilmannii UreBand H. pylori UreAB, confirming that protective immunity againstHelicobacter infections can be elicited by homologous as well asheterologous Helicobacter urease (Dieterich et al. (1999) Inf. Imm. 67,6206-6209).

Recently it has been shown that H. heilmannii does not represent asingle species, but a group of different bacterial species with asimilar spiral morphology, most of which are probably zoonotic inorigin. On the basis of 16S rRNA sequences, “H. heilmannii” has beenclassified into two types (Solnick et al. (1993) J. Infect. Dis. 168,379-385). ‘H. heilmannii’ type 2 organisms are closely related, if notidentical, to the canine and feline Helicobacter spp., namely H. felis,H. bizzozeronii and H. salomonis. More than 50% of the “H. heilmannii”infections in humans however are due to “H. heilmannii” type 1. It isnow accepted that “H. heilmannii” type 1 is identical to “Candidatus H.suis” (O'Rourke et al. (2004) Int J Syst Evol Microbiol. 54, 2203-2212;De Groote et al. (1999) Int. J. Syst Bacteriol. 49, 1769-1777), aspirally shaped bacterium that colonizes the stomach of more than 60% ofslaughterpigs.

Little information is available in the literature on the potential ofvaccine-induced protection against non-pylori helicobacter strains, suchas “Candidatus H. suis”.

In vitro cultivation of “Candidatus H. suis” currently is not possible,but mouse inoculation can be used to grow and maintain this bacteriumviable for more than two years starting from infected pig stomach mucosa(Mendes et al. (1991) cited above; Dick et al. (1989) J. Med. Microbiol.29, 55-62; Park et al. (2003) J. Comp. Pathol. 129:154-160).

SUMMARY OF THE INVENTION

The present invention relates to the use of an antigen preparation of aspecies related to a Candidatus Helicobacter suis for vaccination ofanimals against Helicobacter species, more particularly againstCandidatus H. suis.

A first aspect of the invention thus relates to the use of a compositioncomprising one or more antigen preparations of one or more speciesrelated to Candidatus H. suis for the manufacture of a vaccine againstHelicobacter species, more particularly against Candidatus H. suis. Moreparticularly, the bacterial species related to Candidatus Helicobactersuis envisaged within the context of the present invention is/arespecies of bacteria having a 16S rRNA sequence having at least 93%sequence identity to the sequence of Candidatus Helicobacter suis.According to a particular embodiment the composition used comprises oneor more antigen preparations of one or more species related toCandidatus Helicobacter suis selected from the group consisting ofHelicobacter felis, Helicobacter salomonis, Helicobacter heilmannii(type II), Helicobacter cynograstricus, Helicobacter pylori orHelicobacter bizzozeronii.

According to particular embodiments of the invention the species relatedto Candidatus Helicobacter suis is/are selected from H. felis, H.bizzozeronii or H. cyonograstricus. A further embodiment of theinvention relates the use of an antigen preparation of H. cynogastricusin the preparation of a vaccine against Helicobacter species, moreparticularly against Candidatus H. suis.

A further aspect of the invention thus provides vaccines for use in thevaccination of animals against Helicobacter spp. (species), moreparticularly against Candidatus H. suis. More particularly, the vaccinesof the present invention comprise one or more antigen preparations ofone ore more bacterial species related to Candidatus Helicobacter suisenvisaged within the context of the present invention is/are species ofbacteria having a 16S rRNA sequence having at least 93% sequenceidentity to the sequence of Candidatus Helicobacter suis. According to aparticular embodiment the vaccines comprise one or more antigenpreparations of one or more species related to Candidatus Helicobactersuis selected from the group consisting of Helicobacter felis,Helicobacter salomonis, Helicobacter heilmannii (type II), Helicobactercynograstricus, Helicobacter pylon or Helicobacter bizzozeronii. Thevaccines can further optionally comprise an adjuvant and/or apharmaceutically acceptable carrier.

The vaccines of the present invention can be used for eitherprophylactic or therapeutic immunisation and suitable vaccination routesinclude, but are not limited to, intranasal, subcutaneous andintramuscular immunisation. Suitable vaccination routes also comprisecombination administrations (e.g. oral/intramuscular administration).

According to a particular embodiment the antigen preparation used in thepreparation of a vaccine comprise a lysate of bacteria. Alternativeembodiments include vaccines wherein the antigen preparation compriseswhole-killed bacteria or live-attenuated bacteria. Additionally oralternatively the vaccine according to the invention comprises anantigen preparation which comprises a processed and/or artificialbacterial preparation.

A further aspect of the invention relates to methods of vaccinating ananimal against Helicobacter spp. infection, more particularly against aCandidatus H. suis infection comprising the step of administering acomposition comprising one or more antigen preparations of one or morespecies related to a Candidatus H. suis to the animal, either beforeinfection (for prophylactic vaccination) or after infection has beenidentified (as therapeutic vaccination). Optionally the antigenpreparation is administered together with an adjuvant and/or apharmaceutically acceptable carrier. Specific embodiments of the methodof the invention relate to antigen preparations comprising preparationsof H. felis and/or H. bizzozeronii and/or H. cynogastricus. Morespecifically, the antigen preparation used comprises a lysate of one ormore of these bacteria, but alternative embodiments include antigenpreparations comprising live-attenuated bacteria or processed and/orartificial bacterial preparations. The methods of the invention maycomprise intranasal or subcutaneous administration of the vaccine of theinvention.

Yet a further aspect of the invention relates to an animal model forCandidatus Helicobacter suis infection, more particularly a mouse model.This model allows the in vivo propagation of Candidatus Helicobactersuis in an animal other than its natural host. This model is obtainedaccording to the invention by infecting a mouse with CandidatusHelicobacter suis-infected material. More particularly this comprisesisolating cells from the stomach wall of a pig infected with CandidatusHelicobacter suis, optionally making a homogenate thereof, andintragastrically infecting mice with the cells or homogenate. Theinfection in the mice and the effect of vaccination can be followed upby faecal PCR.

A further aspect of the invention relates to an isolated bacterium ofthe species Helicobacter cynogastricus deposited under Accession NumberLMG P-23100. The invention further relates to the use of Helicobactercynogastricus in the production of a vaccine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described with reference to certainembodiments and to certain Figures but the present invention is notlimited thereto but only by the claims.

The present invention relates to the vaccines and vaccination methodsagainst Helicobacter spp., more particularly against CandidatusHelicobacter suis. The present invention relates to the use of antigensand antigen preparations of certain Helicobacter species for vaccinatinganimals, especially livestock (cows, sheep, horses, . . . ), moreparticularly swine, most particularly cultivated pigs, which areinfected with susceptible to infection with Helicobacter spp., moreparticularly with Candidatus Helicobacter suis. Also other animals, suchas, but not limited to humans, monkeys, rabbits, rodents, cats and dogswhich are suspected to be infected with Helicobacter spp., moreparticularly with Candidatus Helicobacter suis can be vaccinated withthe antigen preparation of the present invention.

“Candidatus Helicobacter suis” as referred to herein is a bacteriumwhich was previously known as “H. heilmannii” type I (Trebesius et al.(2001) J Clin Microbiol. 39, 1510-1516. It is now accepted that “H.heilmannii” type 1 is identical to “Candidatus H. suis” (O'Rourke et al.(2004) Int J Syst Evol Microbiol. 54, 2203-2211; De Groote et al. (1999)Int. J. Syst. Bacteriol. 49, 1769-1777), a spirally shaped bacteriumthat colonizes the stomach of more than 60% of slaughterpigs. CandidatusHelicobacter suis is also defined at the molecular level as theHelicobacter species having a 16S rRNA sequence Genbank Accession AF127028 (D. De Groote et al. (1999) cited above) and AF506788-92(O'Rourke et al. (2004) cited above) and a urease gene sequence asdepicted in Genbank Accession AF508013-AF508014 (O'Rourke et al. (2004)Int J Syst Evol Microbiol. 54, 2203-2211).

The bacterial species suitable for the purpose of the invention arebacteria other than Candidatus Helicobacter suis of the Helicobactergenus, most particularly species that are related to but not identicalto Candidatus Helicobacter suis. The phrase “related to” in the contextof bacterial species is used herein to indicate a phylogenetic relation,preferably expressed by molecular biology parameters. More generally thebacteria related to Candidatus Helicobacter suis have a 16S rRNAsequence which is at least 75%, 80%, 85%, 90%, 93%, 95%, 98% 99%identical with the 16S rRNA sequence of Candidatus Helicobacter suis. Orexpressed in an alternative way, the bacteria related to CandidatusHelicobacter suis comprise an urease coding sequence having a sequencewhich is at least 75%, 80%, 85%, 90%, 95%, 98% 99% identical at the DNAlevel with the urease sequence of Candidatus Helicobacter suis. Speciesthat are identified as ‘closely related to Candidatus Helicobactersuis’, based on the above criteria (with a 16S rRNA sequence having atleast about 93% to 99%, sequence identity with Candidatus Helicobactersuis), can nevertheless be identified as a different species based onother criteria such as, such as, but not limited to, whole-cell proteinprofiles, as described herein. The reference sequence of 16S rRNA ofCandidatus Helicobacter suis corresponds to that of Genbank Accession AF127028.

“Antigen preparation” as used in the context of the present inventionrelates to a composition comprising at least one protein or fragmentthereof which provokes an immune response (hereafter referred to as‘antigen’) when administered to an animal. For use as a vaccine in thecontext of the present invention the antigen preparation may comprisewhole-killed (inactive) bacteria, live-attenuated (weakened) bacteria orprocessed and/or artificial bacterial preparations or combinationsthereof. Processed bacterial preparations included preparations ofbacterial proteins which are partially or completely purified and/orpretreated. Examples of artificial bacterial preparations includeprotein preparations either in part or entirely obtained by synthetic orrecombinant methods.

According to certain embodiments the antigen preparations used forvaccination according to the present invention comprise one or moreantigens obtained from different Helicobacter species which are relatedto Candidatus Helicobacter suis. Examples of suitable antigens includebut are not limited to the urease enzyme, heat shock proteins, cagA andvacA.

A “vaccine” as used herein refers to a composition such as the antigenpreparation described herein which is administered to stimulate animmune response that will protect a person from illness due to thatagent. The vaccine of the present invention is intended for use both asa therapeutic (treatment) vaccine, i.e. for administration to the animalafter infection with the intention to reduce or arrest diseaseprogression and as a preventive (prophylactic) vaccine, foradministration to the animal prior to infection, with the intent toprevent initial (and/or recurrent) infection. The vaccination protectingagainst one species using antigens from another species is referred toas “cross-vaccination” or “heterologous vaccination”.

The present invention is based on the observation that mucosal andparenteral vaccination with heterologous antigens from H. pylori SS1 orH. felis CS1 elicited a significant reduction in bacterial burden butnot sterilizing immunity upon “Candidatus H. suis” challenge.Phylogenetically, H. felis is more closely related to “Candidatus H.suis” than H. pylori (De Groote et al. (1999) cited above), thereforeone could expect to obtain better results with heterologous H. felisimmunization. The present invention demonstrates that only minornon-significant differences in the level of protection between these twogroups are found.

The invention thus relates to heterologous vaccination, i.e. the use ofan antigen preparation of a species related to Candidatus H. suis, toobtain protection against infection by Helicobacter spp., moreparticularly by Candidatus H. suis.

The object of the vaccine and vaccination therewith according to thepresent invention includes obtaining complete protection (sterilisingimmunity) against Helicobacter spp., more particularly againstCandidatus Helicobacter suis in an animal but also reducing thebacterial burden of Helicobacter spp., more particularly of CandidatusHelicobacter suis by at least 25, 40, 60, 80% compared to prior tovaccination and/or compared to animals which have not received thevaccine of the present invention and are/have been subjected to the sameinfectious agent. Most particularly, the present invention relates tovaccines and vaccination strategies which ensure a protective effect orreduced bacterial burden for a prolonged period of time, such as duringat least 4, 6, 10, 12 or more than 12 weeks.

Identification and quantification of such infection and/or bacterialburden in an animal can be done in a number of ways. Classically, thisis done by determining the presence of the infectious agent, or aprotein or DNA sequence thereof in a sample of body fluid or in urine orfaeces. Alternatively, the reaction of the immune system, e.g. thepresence of antibodies to the infectious agent, can be measured.According to a particular embodiment of the invention accurate diagnosisand quantification of Helicobacter infection is obtained byidentification of Candidatus H. suis DNA, e.g. by PCR as described inthe art (Fox and Lee (1997) Lab. Anim. Sci. 47, 222-255). Since“Candidatus H. suis” is hitherto uncultivable, a quantitative ureasetest is use to quantify this species. This assay has been used in theprior art to quantify H. heilmannii, H. felis and H. pylori infection inmouse immunisation studies (Michetti et al. (1994) Gastroenterology 107:1002-1011; Kleanthous (2001) Vaccine 19, 4883-4895; Saldinger et al.(1998) Gastroenterology 115, 891-897). This urease test was found to beless sensitive than determining the number of bacteria aftercultivation.

According to a first aspect, an antigen preparation of one or morespecies related to Candidatus H. suis is used to obtain prophylactic ortherapeutic immunity to Candidatus H. suis. More particularly, theinvention relates to antigen preparations of one or more species havingat least 93% sequence identity in the 16S rRNA or urease proteinsequence to Candidatus H. suis. According to particular embodiments ofthe present invention the species related to Candidatus Helicobactersuis is a/are species selected from the group of H. pyloris, H.bizzozeronii, H. felis and H. Salomonii. Other suitable Helicobacterspecies are H. bilis, H. fenelliae, H. pametensis, H. nemestrinae, H.nemestrinae, H. pametensis, H. acinonychis, H. pullorum, H. mustelae, H.hepaticus, H. cinaedi and H. canis.

The present invention further provides a particular embodiment of astrain useful for the vaccination against Candidatus H. suis, i.e. H.cynogastricus, as described in the Examples herein.

According to a particular embodiment, the antigen preparation is a celllysate, i.e. a mixture obtained upon lysis of bacterial cells. Aparticular example of a bacterial cell lysate is the soluble fraction ofa sonicated bacterial culture, e.g. obtained after filtration.Alternatively or in addition, bacteria can be fragmented using ahigh-pressure homogenizer (e.g. Avestin model EmulsiFlexC5) Optionally,the cell lysate is further inactivated by treatment with formalin, or acomparable agent. Generally not all proteins in a lysate will provoke animmune response. Alternatively, the antigen preparation according to thepresent invention is obtained by fractionation and/or purification ofone or more proteins from a lysate or bacterial culture medium to obtaina composition of enriched or purified antigens. Also falling within theconcept of the present invention are recombinant proteins or fragmentsthereof used as antigen preparation. Most particular examples ofisolated, purified and/or recombinant bacterial proteins suitable in thecontext of the present invention are heat shock proteins and/or ureaseproteins.

The vaccine of the present invention optionally contains only theantigen preparation of the invention. Alternatively, the vaccine cancomprise, in addition to the antigen preparation of the presentinvention, a suitable adjuvant. The type of adjuvant will vary,depending on the type of antigen preparation and rout of administrationused. According to a particular embodiment of the present invention theantigen preparation which is a sonicated antigen solution isadministered intranasally with Cholera toxin (CT) or subcutaneously withsaponine as adjuvant. Any adjuvant known in the art may be used in thevaccine composition, including oil-based adjuvants such as Freund'sComplete Adjuvant and Freund's Incomplete Adjuvant, mycolate-basedadjuvants (e.g., trehalose dimycolate), bacterial lipopolysaccharide(LPS), peptidoglycans (i.e., mureins, mucopeptides, or glycoproteinssuch as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans(e.g., extracted from Klebsiella pneumoniae), streptococcal preparations(e.g., OK432), Biostim™ (e.g., 0 1K2), the “Iscoms” of EP 109 942, EP180 564 and EP 231 039, aluminum hydroxide, saponin, DEAE-dextran,neutral oils (such as miglyol), vegetable oils (such as arachis oil),liposomes, Pluronic® polyols. Adjuvants include, but are not limited to,the RIBI adjuvant system (Ribi Inc.), alum, aluminum hydroxide gel,cholesterol, oil-in water emulsions, water-in-oil emulsions such as,e.g., Freund's complete and incomplete adjuvants, Block co-polymer(CytRx, Atlanta Ga.), SAF-M (Chiron, Emeryville Calif.), AMPHIGEN®adjuvant, saponin, Quil A, QS-21 (Cambridge Biotech Inc., CambridgeMass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.) orother saponin fractions, monophosphoryl lipid A, Avridine lipid-amineadjuvant, heat-labile enterotoxin from E. coli (recombinant orotherwise), cholera toxin, or muramyl dipeptide, among many others.According to a particular embodiment recombinant mutant of Escherichiacoli heat-labile toxin is added to the antigen preparation prior toinjection into the animal.

According to another aspect, the present invention relates to the use ofthe vaccines of the present invention to obtain prophylactic ortherapeutic immunity to Helicobacter spp. such as those referred toherein, more particularly to Candidatus H. suis.

According to a first embodiment the invention provides antigenpreparations for use in prophylactic vaccination which ensure protectionagainst Helicobacter spp., more particularly against Candidatus H. suiswhich is more than transient. Transient infection of prophylacticallyimmunised mice has only been reported once in the H. pylori model(Garhart et al. 2002, Infect. Immun. 70:3529-3538). The presentinvention shows the evolution of protection over time. This is performedby a method for detection of infection in faecal samples, particularlydeveloped for this purpose. A PCR is carried out on faecal samplescollected at subsequent time points, which gives an impression of thecolonisation in the stomach with “Candidatus H. suis”. The PCR reactionis performed on a small fragment of the 16S rRNA gene. Typically, thisfragment has a length of less than 400 bp (e.g. a fragment between 200and 400 bp, a fragment between 200 and 100 bp or a fragment between 100and 50 bp), more particularly a fragment which comprises sequences forPCR amplification that are species-specific. This allows the detectionof degraded 16S rRNA of a specific Helicobacter species in faecalsamples. Larger fragments or full length 16S rRNA, such as detected ingastric samples (De Grootte et al (2000) cited above), could not bedetected in faecal samples of pigs. It is demonstrated herein that thereis a decrease in excretion of Helicobacter DNA from one week afterinfection in the immunised mice compared to the non-immunised mice, andthat colonization in immunised mice never reaches the same level as innon-immunised mice.

In another aspect the present invention relates to methods fortherapeutic immunization, when the organisms have already orientated thehost immune response to their benefit.

The antigen preparations or vaccines of the present invention can beadministered via any suitable route, such as by mucosal (intranasal),perenteral, or intramuscular administration, oral, intradermal,intraperitoneal, intravenous, or subcutaneous administration. Suitablevaccination routes also comprise combination administrations (e.g.oral/intramuscular administration). According to a specific embodimentof the invention therapeutic immunization is performed by parenteraladministration of the antigen preparation of the invention. Parenteralimmunization can mobilize cells from systemic origin that have not beenalready primed in one given direction by a Helicobacter infection (Guyet al. (1999) Vaccine 17, 1130-1135). According to another specificembodiment of the invention, intramuscular administration is used forefficient vaccination.

The antigens the present invention can be administered alone or withsuitable pharmaceutical carriers, and can be in solid or liquid formsuch as, tablets, capsules, powders, solutions, suspensions, oremulsions.

The solid unit dosage forms can be of the conventional type. The solidform can be a capsule, such as an ordinary gelatin type containing theproteins or peptides of the present invention or the antibodies orbinding portions thereof of the present invention and a carrier, forexample, lubricants and inert fillers such as, lactose, sucrose, orcornstarch. In another embodiment, these compounds are tableted withconventional tablet bases such as lactose, sucrose, or corn starch incombination with binders like acacia, corn starch, or gelatin,disintegrating agents such as, corn starch, potato starch, or alginicacid, and a lubricant like stearic acid or magnesium stearate.

The antigens of the present invention may also be administered ininjectable dosages by solution or suspension of these materials in aphysiologically acceptable diluent with a pharmaceutical carrier. Suchcarriers include sterile liquids such as water and oils, with or withoutthe addition of a surfactant and other pharmaceutically acceptableadjuvants. Illustrative oils are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, or mineraloil. In general, water, saline, aqueous dextrose and related sugarsolution, and glycols such as, propylene glycol or polyethylene glycol,are preferred liquid carriers, particularly for injectable solutions.

For use as aerosols, the antigens of the present invention in solutionor suspension may be packaged in a pressurised aerosol containertogether with suitable propellants, for example, hydrocarbon propellantslike propane, butane, or isobutane with conventional adjuvants. Thematerials of the present invention also may be administered in anon-pressurised form such as in a nebulizer or atomizer.

According to yet another aspect of the invention, an in vivo animalmodel is provided for infection with Candidatus H. Suis. The inventionprovides a method of obtaining Candidatus H. suis infection in alaboratory or model animal, such as mice, which method comprisesintragastrically inoculating the laboratory or model animal withhomogenates of cells obtained from the stomach wall of infected animals,more particularly infected pigs. According to a specific embodiment theupper cell layers and mucus are scraped from the antrum and homogenized.According to yet a further particular embodiment the material from thestomach wall of infected animals is homogenized in lyophilisation medium(2 volumes of horse serum, 1 volume of BHI broth and 10% glucose) (LYM).Optionally, larger particles are removed by centrifugation. According toa particular embodiment the stomach from infected laboratory animal ormodel animal so obtained is again dissected and homogenized for at leasttwo additional passages in the same animal. The infection is optionallyfollowed up by faecal PCR as described herein.

FIGURE LEGENDS

The following Figures represent illustrative embodiments of theinvention.

FIG. 1: Quantitative urease assay on gastric stomach tissue of differentsites (cardia, fundus, antrum) from pigs infected with CandidatusHelicobacter suis from mice (n=5) and controls (n=5) according to oneembodiment of the invention.

FIG. 2: Excretion of “Candidatus Helicobacter suis” DNA in faeces ofBALB/c mice immunised intranasally with H. pylori antigens (♦) or H.felis antigens (⋄) compared to unimmunised mice (▴), 1 to 16 weeks afterinfection with “Candidatus Helicobacter suis”, according to oneembodiment of the invention. The excretion is expressed as percentage ofmice positive in PCR per group.

FIG. 3: Excretion of “Candidatus Helicobacter suis ” DNA in faeces ofBALB/c mice immunised subcutaneously with H. pylori (▴) or H. felis (⋄)antigens, compared to unimmunised (▴) animals, 1 to 16 weeks afterinfection with “Candidatus Helicobacter suis”, according to oneembodiment of the invention. The excretion is expressed as percentage ofmice positive in PCR per group.

FIG. 4: Quantitative urease activity of gastric stomach tissue,represented as OD value (550 nm), from mice intranasally immunised withH. pylon or H. felis antigens according to one embodiment of theinvention. Solid lines represent the geometric mean for each groupstudied. (Cs) animals challenge infected with “Candidatus Helicobactersuis”; (Cp) animals challenge infected with H. pylori; (Cf) animalschallenge infected with H. felis; (IN,p) intranasal immunisation with H.pylori antigens; (IN,f) intranasal immunisation with H. felis antigens.

FIG. 5: Quantitative urease activity of gastric stomach tissue,represented as OD value (550 nm), from mice subcutaneously immunisedwith H. pylori (SCp) or H. felis (SCf) antigens according to oneembodiment of the invention. Solid lines represent the geometric meanfor each group studied. Significant differences (P<0.05) betweenimmunised and non-immunised challenged animals, for each Helicobactersp., are indicated with letter a. (Cs) animals challenge infected with“Candidatus Helicobacter suis”.

FIG. 6: Excretion of “Candidatus Helicobacter suis” DNA in the faeces ofBALB/c mice immunised intranasally with H. felis CS1 or H. bizzozeroniiantigens three weeks post “Candidatus Helicobacter suis” challengeaccording to particular embodiments of the invention. The excretion isexpressed as percentage of mice positive in PCR. The differences betweenthe experimental conditions are explained in detail in Table 1.

FIG. 7: Quantitative urease activity of gastric stomach tissue,represented as OD value (550 nm). Solid lines represent geometric meanfor each group studied. A significant (P<0.05) decrease in ureaseactivity between nonimmunised, challenged (group 9) and immunisedanimals was found for group 1 and group 2, both representing intranasalimmunization with H. felis CS1 sonicated antigen solution or H.bizzozeronii respectively (a), according to particular embodiments ofthe invention. The differences between the experimental conditions areexplained in detail in Table 1.

FIG. 8A: Serumconversion (s/p values) against H. felis antigens aftervaccination with H. felis antigens (serology data in swine) (group 1: ▪;group 2: ♦; group 3: ▴) and H. bizzozeronii antigens (group 4: ●)(adjuvans only: _) Pre: pre immunisation; 1×: 3 weeks after the firstimmunisation; 2×: two weeks after the second immunisation.

FIG. 8B: Serumconversion (s/p values) against H. bizzozeronii antigensafter vaccination with H. felis antigens (group 3: ▴) and H.bizzozeronii antigens (group 4: ●) (adjuvans only: _) Pre:pre-immunisation; 1×: 3 weeks after the first immunisation; 2×: twoweeks after the second immunisation.

FIG. 9A: Dendrogram (A) derived from the numerical analysis of thewhole-cell protein profiles (B) of H. cynogastricus, H. pylori, H.bizzozeronii, H. salomonis and H. felis reference strains.

FIG. 9B: Similarity matrix based on 16S rRNA sequence comparison.

FIG. 10: Phylogenetic tree for 25 strains of Helicobacter species basedon 16S rRNA sequence similarity. The scale bar represents a 10%difference in nucleotide sequences as determined by measuring thelengths of the horizontal lines connecting any two species.

FIG. 11: Genomic sequence of 16S rRNA gene of Helicobactercynogastricus.

The invention is illustrated but not limited to the following examples.

EXAMPLES

General Methodology.

Mice. All experiments involving animals were approved by the Animal Careand Ethics Committee of the Faculty of Veterinary Medicine, GhentUniversity. Five week-old male, SPF BALB/c mice were purchased from anauthorized breeder (HARLAN, Horst, The Netherlands). The animals werehoused individually in autoclaved filter top cages and provided with acommercial diet (TEKLAD, HARLAN) and water ad libitum. After anadaptation period of one week, the animals were used in the experiments.

Antigens for vaccination. H. pylori SS1, H. felis CS1 (ATCC 49179) andH. bizzozeronii (CCUG 35545) were grown on brain heart infusion (BHI,Oxoid, England) agar plates supplemented with 10% horse blood, 5 mg/mlamphotericin B, 10 mg/ml vancomycin, 5 mg/ml trimethoprim lactate and2500 units/I polymyxin B (Skirrow, Campylobacter Selective Supplement,Oxoid) and Vitox supplement (Oxoid). Plates were incubated at 37° C. inmicro aerobic conditions. The antigens used for immunisation wereprepared by harvesting 3-day old cultures in sterile phosphate bufferedsaline. The bacterial suspension was sonicated (8 times 30 seconds, 50%capacity; Misonix, Incorporated, USA). After centrifugation (5,000 g, 5min., 4° C.) the supernatant was filtered through a 0.22-μm pore filter(Schleisser Schuell, Dassel, Germany) and stored at −70° C. Afterwards,protein concentration was determined by the Lowry assay (Lowry et al.(1951) J. Biol. Chem. 193, 265-275).

Formalin inactivated bacterial cultures were prepared by transferringbacterial cultures from agar plates to BHI broth supplemented with 0.2%Skirrow, 0.6% Vittox and 10% horse serum. After 24 h of incubation at37° C. 0.5% formaldehyde was added and further incubated at 37° C.Twenty-four hours later the culture was cooled to 4° C. and checkedmicroscopically for presence of intact bacteria. Twenty percent ofsodiumbisulphite 0.166M was added to neutralize formaldehyde.Afterwards, protein concentration was determined by the Lowry assay.

Intranasal immunisation. Forty-five mice were divided into seven groupsof six animals (groups 1-7) and one group of three animals (group 8).All animals from groups 1 and 3 were immunised intranasally with H.felis CS1 and those of groups 2 and 4 with H. pylori SS1, twice withthree weeks time interval. Intranasal immunisation was done by applyingabout 100 μl with 100 μg of sonicated antigen solution mixed with 5 μgcholera toxin (List, Campbell, Calif., US) on the external nares ofunanaesthetized mice. Mice from groups 5, 6, 7 and 8 were not immunised.Four weeks after the final immunisation, all animals from groups 2 and 5and all animals from group 1 and 6 were challenged with H. pylori or H.felis respectively, by intragastric inoculation with 0.3 ml of thebacterial suspension. This homologous vaccination experiment serves as acontrol. At the same time all animals from groups 3, 4 and 7 wereinoculated intragastrically with “Candidatus H. suis”. For this purposea frozen stock from “Candidatus H. suis” was placed at 37° C. for 15minutes.

During 1, 2, 4, 5, 6, 7, 9, 11, 13 and 15 weeks after challenge, faecalmaterial was collected for three consecutive days from each individualmouse inoculated with “Candidatus H. suis” to screen for the presence ofbacterial DNA. PCR on faecal samples was performed as described below.

Sixteen weeks after challenge, all animals were euthanized by cervicaldislocation following isoflurane anaesthesia (IsoFLo, Abbot, Ill., US).From all animals, half of the stomach was used for a quantitativeurease-test (Corthésy-Theulaz et al. (1995) cited above) as describedbelow. From the other half, 2 mm² tissue samples from the fundic regionwere frozen (−20°) and used for PCR specific for “Candidatus H. suis”(samples from group 3, 4 and 7), H. felis (samples from groups 1 and 6)or H. pylori (samples from groups 2 and 5) as described below.

Subcutaneous immunisation. Twenty-one mice were divided into threegroups of six animals (groups 1-3) and one group of three animals (group4). Animals from groups 1 and 2 were immunised with H. pylori or H.felis respectively, three times with three weeks time interval. For thispurpose about 100 μl with 100 μg of the sonicated bacterial antigensolution was mixed in equal amounts with saponine adjuvant and injectedsubcutaneously at the lower back of the animals. Four weeks after thefinal immunisation, animals from groups 1, 2 and 3 were infected with“Candidatus H. suis” as described in protocol 1. Animals from group 4were not immunised or challenged. Sampling of faecal material, fromgroups 1, 2 and 3, during the experiment and sampling of stomachmaterial from all animals, at the end of the study, was done asdescribed in protocol 1.

Preparation of Candidatus H. suis in an in vivo system, for use inchallenge experiments. Thirty pig stomachs were obtained from theslaughterhouse. The stomachs were opened and the remaining food wasrinsed off with autoclaved tap water (37° C.). A small mucosal fragmentfrom the antrum (1 cm from the torus pyloricus) was taken to screen forthe presence of “Candidatus H. suis”. Half of this fragment was used forrapid urease test (CUT, Temmler Pharma, Marburg, Germany, 37° C. for 1h). The other half was frozen (−20° C.) and used for specific detectionof “Candidatus H. suis” by PCR (De Groote et al. (200) J. Clin.Microbiol. 38, 1131-1135) as confirmation for the urease test. Stomachsfrom which the urease test give the quickest positive results wereprocessed in the first instance. Therefore upper cell layers and mucusfrom the antrum were scraped. Scrapings were homogenized inlyophilisation medium (2 volumes of horse serum, 1 volume of BHI brothand 10% glucose) (LYM). The homogenate was then centrifuged (5000 g, 5min) to remove large particles. Supernatant was diluted 1/10 in LYM andintragastrically inoculated in three BALB/c mice. Two weeks later, thesemice were euthanized, the stomachs were emptied and a fundus tissuesample was taken for rapid urease test. Urease positive stomachs werehomogenized in LYM (5 ml LYM/stomach). After this first passage, twoextra mouse passages were performed. Finally, infected mouse stomachhomogenate from 15 mice was frozen at −70° C.

Preparation of H. pylori and H. felis for use in challenge experiments.H. pylori SS1 or H. felis CS1 were grown on BHI agar plates, staticallyat 37° C. in micro-aerobic conditions. After 3 days, bacteria wereharvested, transferred to BHI broth supplemented with 0.2% Skirrow, 0.6%Vittox and 10% horse serum, and incubated statically at 37° C. inmicro-aerobic conditions for 24 h. A bacterial suspension with anabsorbance of 1.5 (450 nm) and an absorbance of 1.5 (660 nm) for H.pylori and H. felis respectively were consequently prepared in BHIbroth, corresponding to approximately 10⁷ cfu/ml for H. pylori and 10⁸cfu/ml for H. felis as confirmed by titration.

Statistical analysis. The presence of bacteria in faeces, as determinedby PCR, was compared between the treatment groups by a generalised mixedmodel with PCR-positivity as binary response variable, time andtreatment as categorical fixed effects and mouse as random effect.Pairwise comparisons were performed between the non-immunised group andthe H. pylori and H. felis immunised groups at a global significancelevel of 5%, and a comparison wise significance level of 1.3% (adjustedby Bonferroni's technique with 3 comparisons).

The quantitative urease tests were compared by a fixed effects modelwith “OD value” as response variable and “treatment group” as fixedeffect. Pairwise comparisons were performed between the treatment groupsat a global significance level of 5% with again using Bonferroni'stechnique for multiple comparisons.

The quantitative urease tests shown in FIG. 7 were compared between thetreatment groups by a Students t-test.

Example 1 Experimental Infection of Pigs with Candidatus H. suis

Five-week-old pigs (n=10) were purchased from a specific pathogen free(SPF) breeding unit negative for Candidatus H. suis and randomly dividedin a control group (group A) and an infected group (group B) with 5 pigsin each group. After an adaptation period of 1 week the pigs were usedin the experiment. Before inoculation with the pathogen, all pigs weretreated intramuscularly with 60 mg/kg cimetidine to reduce stomach acidproduction and were anaesthetised. Group A was the control group and wassham inoculated (inoculum from urease negative stomachs fromnon-infected BALB/c mice) on day 7, day 14 and day 21. Group B wasinoculated with Candidatus H. suis (a stomach homogenate of CandidatusH. suis infected mice) on day 7, day 14 and day 21. Immediately beforeand immediately after administration of the murine stomach homogenate,pigs were intragastrically inoculated with Brucella Broth (BectonDickinson, Erembodegem, Belgium) supplemented with 10% fetal bovineserum and 0.75% agar, to delay the passage of the bacterial suspensionthrough the duodenum. Prolonged exposure of the gastric mucosa to thebacteria is assumed to make the stomach more susceptible to colonisationwith Helicobacter bacteria.

All animals were euthanized 5 weeks after the third inoculation, andnecropsied immediately. At necropsy, the stomachs were excised. Themucosal surface from the pars oesophagea was macroscopically examinedand lesions were scored on a scale of 0-5 using the method of Hessing etal. (1992). (score 0=intact mucosa, score 1=mild hyperkeratosis (<50%surface area), score 2=severe hyperkeratosis (50% or more of surfacearea), score 3=hyperkeratosis and a few small erosions (less than 5 andshorter than 2.5 cm), score 4=hyperkeratosis and extensive erosions(more than 5 erosions and/or longer than 2.5 cm), score 5=hyperkeratosisand very large erosions (more than 10 erosions or longer than 5 cm)and/or ulcers.

In group B, inoculated with “Candidatus H. suis”, three animals had alesion score of 3, one animal a score of 4 and one animal a score of 5.In the control group, lesion scores were 2 (one animal), 3 (two animals)and 4 (two animals). The difference in lesion scores between “CandidatusH. suis” infected animals and control animals was not significant(P=0.36).

After scoring, three sites from the glandular mucosa (0.5 cm²) from eachstomach were sampled for PCR, quantitative urease test and histology.These three sites corresponded to the cardia (immediately adjacent tothe margo plicatus), the fundus, and the antrum pyloricum (1 cm awayfrom the torus pyloricus).

PCR for specific detection of “Candidatus H. suis” infection in gastrictissue. DNA was extracted with DNeasy Tissue Kit (Qiagen, Hilden,Germany). PCR for specific detection of “Candidatus H. suis” wasperformed as described by De Groote et al. (2000) cited above. Thesamples of all control animals were negative. All fundus and antrumsamples and 3/5 cardia samples of group B were positive in PCR.

The Urease assay was performed as described by Corthésy-Theulaz et al.(1995) cited above. Mean OD-values for each sampling site are shown inFIG. 1. Significant differences (P<0.05) between control animals andanimals infected with “Candidatus H. suis” were found for all tissuesamples originating from the fundus and the antrum (cardia, P=0.0676;fundus, P=0.0038; antrum pyloricum P=0.0011). There was no significantdifference in mean OD value for the different sampling sites in infectedanimals (cardia vs. fundus P=0.2280; cardia vs. antrum pyloricumP=0.1733 and fundus vs. antrum pyloricum P=0.7824).

Histological examination was performed using a polyclonal goat anti-H.pylori antibody as described by De Groote et al. (2000). Animalspositive for “Candidatus H. suis” had lesions predominantly in theantral mucosa. In this stomach region, focal lymphoplasmacytic cellularinfiltrates in the mucosa were present in all 5 animals. In 3/5 infectedanimals antral lymphoid follicles with germinal centers were present.Due to the size of the follicles, they were associated with displacementand loss of gastric glands . In 2/5 infected animals no follicles couldbe detected in the antral mucosa, but these animals did show aggregatesof lymphocytes and plasmacells in the lamina propria. In the fundus of“Candidatus H. suis” infected animals, only mild scattered infiltrationof lymphocytes was present. In the antrum, colonization was present inthe mucus overlying the surface epithelium and in the surface foveola,but did not extend deep into the gastric pits. Colonization of thefundic mucosa with “Candidatus Helicobacter suis” was detected in theglandular foveola and extended down halfway the gastric pits. Bacteriawere found in close contact with gastric epithelial cells i.e. mucusproducing cells and parietal cells. No bacteria were demonstrated in thestomach of the control animals. Control animals are negative for“Candidatus H. suis” (Group A).

Example 2 Experimental Set-Up of Comparative Immunization with SpeciesRelated to Candidatus H. suis

In a parallel experiment, the effect of immunization with H. felis or H.bizzozeronii sonicate, H. bizzozeronii or H. felis formaline inactivatedand control adjuvants was tested using intra-nasal or subcutaneousimmunization routes. Sixty mice were divided into ten groups of sixanimals as shown below (Table 1). TABLE 1 Experimental design AntigenGroup^(a) preparation^(b) Adjuvant^(c) Route^(d) Challenge 1 H. felissonicate CT IN “Candidatus H. suis” 2 H. bizzozeronii CT IN “CandidatusH. suis sonicate 3 H. bizzozeronii CT IN “Candidatus H. suis” formalineinactivated 4 H. felis sonicate saponine SC “Candidatus H. suis” 5 H.felis sonicate saponine + SC “Candidatus H. suis” LT 6 H. felissaponine + SC “Candidatus H. suis” formaline LT inactivated 7 H.bizzozeroni saponine + SC “Candidatus H. suis” sonicate LT 8 /saponine + SC “Candidatus H. suis” LT 9 / / / “Candidatus H. suis” 10 // / /^(a)Mice were divided into 10 experimental groups (1-10).^(b)One hundred micrograms of sonicated antigen solution or formolinactivated antigens were used for each immunisation.^(c)Five μg of cholera toxin was used for the intranasal immunisation.For the subcutaneous immunisation, antigens in solution was mixed withequal amount of saponine adjuvant and 1 μg of LT.^(d)IN, intranasally; SC, subcutaneously.

Groups 1, 2 and 3 were immunised intranasally twice with three weeksinterval. Therefore 100 μg of H. felis or H. bizzozeronii sonicatedproteins, mixed with 5 μg of cholera toxin (List, Campbell, Calif., US),was applied on the external nares of unanaesthetized mice.

Groups 4-7 were immunised subcutaneously three times with three weekstime interval. For this purpose 100 μg of the sonicated antigen solutionwas mixed in equal amounts with saponine adjuvant. Immediately prior toinjection 1 μg of recombinant mutant of Escherichia coli heat-labiletoxin (LTR192G, donated by J. Clements) was added and then the antigenpreparation was injected subcutaneously at the lower back of theanimals.

Animals from group 8 were immunised subcutaneously with saponineadjuvant plus LTR192G only. Mice from groups 9 and 10 were notimmunised.

Four weeks after the final immunisation, a frozen stock from “CandidatusH. suis”, was placed at 37° C. for 15 minutes. All animals from groups1-9 were challenged by intragastric inoculation with 0.3 ml of the“Candidatus H. suis” stock.

During the third week after challenge, faecal material was collected forfour consecutive days from each individual mouse to screen for thepresence of “Candidatus H. suis” DNA. PCR on faecal samples wasperformed as described below.

Six weeks after challenge, all animals were euthanized by cervicaldislocation following isoflurane anaesthesia (IsoFLo, Abbot, Ill., US).From all animals, half of the stomach was used for a quantitativeurease-test (Corthésy-Theulaz et al. 1995, above) as described below.From the other half, 2 mm² tissue samples from the fundic region werefrozen (−20°) and used for PCR specific for Candidatus H. suis asdescribed below.

Example 3 Faecal Excretion of “Candidatus H. suis” DNA in Faeces ofNon-Immunised and Immunised Mice

Detection of ‘Candidatus H. suis’ DNA in Faecal Samples.

PCR on faecal samples was performed to evaluate the excretion of“Candidatus Helicobacter suis” DNA. DNA was extracted using QIAamp® DNAStool Mini Kit (Qiagen, Hilden, Germany). Primers HS 586gggaggacaagtcaggtgtgaa [SEQ ID:1] and HS641 tctcccacactccagaaggatag [SEQID:2], complementary to the 16S rRNA genes from “Candidatus Helicobactersuis” were used to amplify a 79-bp fragment. The specificity of theprimerset was tested on DNA extracts from 17 other Helicobacter speciesand from Campylobacter jejuni (table 2). The sensitivity was assayed byadding cloned 16S rRNA to a fecal control sample. After DNA isolationand PCR, a fragment was obtained when about 100,000 copies were added toa control sample. TABLE 2 ^(b)Strain Species CCUG 38995 H. bilis CCUG29260 H. pametensis CCUG 32350 H. nemestrinae NCTC 11961 H. pylori LMG6444 C. jejuni LMG 16318 H. pullorum LMG 18044 H. mustelae LMG 16316 H.hepaticus LMG 18086 H. canis LMG 11759 H. fenelliae LMG 14378 H.nemestrinae LMG 12678 H. pametensis LMG 12684 H. acinonychis R 10t51 H.bizzozeronii R 1053 H. salomonis R 3647 H. felis LMG 7543 H. cinaedi^(b)Bacterial strains used for the evaluation of the Candidatus“Helicobacter suis” specific PCR on faecal samples.

PCR reaction mixtures (25 μl) contained 50 pmole of each primer(Invitrogen Life Technologies, Merelbeke, Belgium), 200 μM of eachdeoxynucleoside triphosphate (Amersham Pharmacia Biotech, Puurs,Belgium), 0.03 U/μl Taq platinum, 1.5 mM MgCl₂ and 1× PCR buffer(Invitrogen Life Technologies). PCR products were run on 1.5% agarosegels containing 50 ng/ml ethidium bromide. After 1 hour at 160V theproducts were visualized with an UV transilluminator. The CandidatusHelicobacter suis amplified fragment has a length of 80 bp. No PCRfragments are obtained using any of the other species presented in table2.

Statistical Analysis

The presence of bacteria in faeces, as determined by PCR, was comparedbetween the treatment groups by a generalised mixed model withPCR-positivity as binary response variable, time and treatment ascategorical fixed effects and mouse as random effect. Pairwisecomparisons were performed between the non-immunised group and the H.pylori and H. felis immunised groups at a global significance level of5%, and a comparison wise significance level of 1.3% (adjusted byBonferroni's technique with 3 comparisons).

The presence of DNA in faecal samples as determined by PCR assay, pergroup, and calculated for each week of sampling is depicted in FIG. 2and FIG. 3 for experiment 1 and experiment 2 respectively. There was anoverall significant difference in faecal excretion between non-immunisedand intranasally immunised animals both for H. pylori and for H. felissonicated antigen solution (P<0.0001) in that the intranasally immunisedmice excreted less “Candidatus Helicobacter suis” DNA in the faeces incomparison to the non-immunised “Candidatus H. suis” challenged animals.The difference in excretion between the two intranasally immunisedgroups was not significant (P=0.0241).

A significant difference was found between non-immunised mice and miceimmunised subcutaneously with H. pylori sonicated antigen solution(P=0.0001). The difference between non-immunised mice and mice immunisedsubcutaneously with H. felis sonicated antigen solution was significantat P=0.0175. There was no significant difference between the twosubcutaneously immunised groups (P=0.2445).

The results of the experiment, described in table 1, on the number ofmice excreting “Candidatus Helicobacter suis” DNA in the faeces threeweeks post challenge are shown in FIG. 6. Intranasal immunisation causeda lower excretion compared to the non-immunised “Candidatus Helicobactersuis” challenged animals. For the subcutaneously immunised groups aneffect of immunisation was only detectable in group 4, representinganimals immunised with H. felis CS1 sonicated antigen solution.

Example 4 Quantitative Urease in Gastric Tissue

Quantitative Urease Test of Gastric Tissue.

The stomach sample was immersed in 500 μl of CUTest and incubated at 37°C. for 3 hours. After centrifugation (5 min, 100 g) the supernatant wasused for spectrophotometric quantification at an OD of 550 nm. The assaywas performed as described in Corthésy-Theulaz et al. (1995)Gastroenterol. 109, 115-121).

For the homologous intranasal immunisation (FIG. 4) there was asignificant difference (P<0.05) in urease activity between non-immunisedand immunised animals both for H. pylori SS1 and H. felis CS1.Immunization of animals with H. pylori SS1 or H. felis CS1 sonicatesantigen solution before “Candidatus H. suis” challenge resulted in asignificant decrease (P<0.0001) in urease activity compared to thenon-immunised challenged group.

Subcutaneous immunisation (FIG. 5) with H. pylori or H. felis antigens,resulted in a significant (P<0.0001) decrease in urease activitycompared to the non-immunised challenged group.

In the experiment described in Table 1, the mean OD value fromnon-immunised non-challenged mice was 0.103. The mean OD value fromnon-immunised mice, challenged with “Candidatus Helicobacter suis” was2.038. A significant (P<0.05) difference in urease activity betweennonimmunised, challenged (group 9) and immunised animals was found forgroup 1 and group 2, representing intranasal immunization with H. felisCS1 or H. bizzozeronii sonicated antigen solution respectively. None ofthe subcutaneously immunised groups showed a significant decrease inurease activity compared with group 9. In the statistical analysis,group 7, representing animals immunised subcutaneously with H.bizzozeronii sonicated antigen solution, showed the lowest P value(P=0.085).

Example 5 PCR Analysis of Gastric Tissue

PCR Analysis of Gastric Tissue.

DNA of the stomach sample was extracted with DNeasy Tissue Kit (Qiagen).PCR for detection of “Candidatus H. suis”, H. felis or H. pylori wereperformed as described previously (De Groote et al. (2000) cited aboveand De Groote (2001) J. Clin. Microbiol. 39, 1197-1199).

None of the animals immunised intranasally with H. felis CS1 sonicatedantigen solution, followed by H. felis CS1 challenge containedCandidatus Helicobacter suis DNA. In contrast, immunization with H.pylori SS1 and homologous challenge all showed the presence ofCandidatus Helicobacter suis DNA in stomach samples. After heterologousintranasal and subcutaneous immunisation of mice with H. pylori or H.felis antigens, and challenged with “Candidatus H. suis”, all stomachsamples contained Candidatus Helicobacter suis DNA.

In the experiment described in Table 1, all animals from group 10 werenegative in the PCR test specific for “Candidatus Helicobacter suis”.From the challenged animals only one animal from group 7 was negative inPCR test.

Example 6 Seroconversion in Pigs Upon Immunization with H. felis or H.bizzozeronii

30 conventional pigs of 5 weeks old (Agrivet Merelbeke), were dividedinto groups of 6 animals. All groups were immunised twiceintramuscularly, with a three week interval, using 0.5 mg of antigens(bacterial lysate) (group 1-4) or using adjuvans only (5). Preliminaryexperiments with 0.1, 0.5 and 1 mg of antigen preparation showed that adosis of 0.5 mg provoked the largest immune response. TABLE 3Immunisation scheme for serumconversion Group Antigen preparationSpecies additives 1 0.5 mg sonicated bacterial H. felis* lysate 2 0.5 mgsonicated bacterial H. felis CT (Cholera lysate Toxin) 3 0.5 mgsonicated bacterial H. felis CT lysate (formol inactivated) 4 0.5 mgsonicated bacterial H. bizzozeronii CT lysate 5 Adjuvans only CT*H felis strain ATCC 49179

Preserum was collected from all animals. Two weeks after the firstimmunisation, prior to the second immunisation, blood was collected.Blood was further collected after one, two and three weeks after thesecond immunisation.

ELISA plates were coated with bacterial lysate and used for incubationwith serum. Bound antigens were detected with Alkaline phoshate labelledpolyclonal goat anti swine antibodies.

The results are depicted in FIG. 8 a and 8 b wherein ELISA results aredepicted for plates respectively coated with H. felis and H.bizzozeronii.

Using ELISA plates coated with H. felis, serumconversion could only bedemonstrated in mice immunised with H. felis antigens. With platescoated with H. bizzozeronii serumconversion could be demonstratedagainst H. bizzozeronii.

Example 7 Identification of Helicobacter cynogastricus

A Helicobacter species related to Candidatus H. suis and suitable forthe preparation a vaccine is a novel Helicobacter species Helicobactercynogastricus (also designated in the present invention as strainJKM4^(T)), which is a Gram-negative, microaerophilic helical-shaped rod.

Isolation of JKM4T

A Helicobacter strain (JKM4^(T)) was isolated from the antrum and fundusregion of the stomach of a euthanized dog at the Faculty of VeterinaryMedicine, Ghent University, Belgium. Samples were handled as describedby Gruntar et al. (2003) Int J Med Microbiol 293, 65. Bacteria weregrown on brain heart infusion agar (BHI; Oxoid, Ltd., Basingstoke,England), containing 10% (vol/vol) horse blood, 5 mg/l amphotericin B(Fungizone; Bristol-Myers Squibb, Epernon, France), Campylobacterselective supplement (Skirrow, Oxoid; containing 10 mg/l vancomycin, 5mg/l trimethoprim lactate and 2500 U/l polymyxin B), and Vitoxsupplement (Oxoid). Plates were incubated with lids, uppermost at 37°C., under humidified micro-aerobic conditions in a closed circuit,created by evacuating 80% of the normal atmosphere and introducing a gasmixture of 8% CO₂, 8% H₂ and 84% N₂.

Plates were checked every two days and BHI broth was added on the agarsurface to ensure plates would not dry up. Primary growth occurred after10 days of incubation as an oily aspect on the broth covering the agarmedium. Light microscopical examination of the broth revealed thepresence of spiral-shaped, motile organisms. Gram staining proved theGram negativity and the helical shape of the isolate. Bacterial growthof subcultures occurred as a spreading layer on moist agar plates.Pinpoint colonies were observed when an abundant amount of bacteria wasbrought on a dry agar surface. Bacteria grown on a dry agar mostly losttheir spiral morphology and transformed to coccoid forms.

Bacteria with typical spiral morphology were harvested in BHI broth andstored at −70° C. in a medium consisting of 7.5 g glucose, 25 ml BHI(Oxoid) and 75 ml sterile inactivated horse serum. The isolated strainJKM4T is a novel helicobacter species, Helicobacter cynogastricus (seebelow) and has been deposited on Jun. 6, 2005 with Accession Number LMGP23-100 at the Belgian Coordinated Collections of Micro-organisms(BCCM™/LMG) [Laboratorium voor Microbiologie, Universiteit Gent (RUG),K. L. Ledeganckstraat 35, 9000 Gent, Belgium] by Katleen van den Buick.

Phenotypic Studies

For scanning and transmission electron microscopic examination,bacterial cultures were fixed in 2.5% glutaraldehyde and 2%paraformaldehyde in cacodylate buffer (0.1 M, pH 7.3). They werepostfixed in 1% (w/vol) osmium tetroxide in distilled water. Samples forscanning electron microscopy were dehydrated in alcohol and acetone forsubsequent critical point drying in liquid carbon dioxide, glued withcarbon cement on aluminium stubs, sputtered with a gold layer andexamined with a Philips 501 SEM. Samples for transmission electronmicroscopy were block stained with 2% (wt/vol) uranyl acetate indistilled water and dehydrated in ethanol. They were embedded inEpon-Spurr's (1:1) medium. Ultrathin sections were cut from samples inwhich bacteria were demonstrated, stained with lead citrate and examinedwith a Philips EM 208S. Biochemical and tolerance tests were carried outas recommended by Dewhirst et al. (2000) Int J Syst Evol Microbiol 50,2231-2237, for the description of new species of the genus Helicobacter.The isolate was tested for oxidase, catalase (with 3% hydrogen peroxide)and rapid urease activity, and for hydrolysis of indoxyl acetate. Thebacteria were also subjected to API Campy test strips (Biomerieux SA,Marcy-I'Etoile, France), which include tests for urease activity,nitrate reductase activity, esterase activity, hippurate hydrolysis,gamma-glutamyl transpeptidase activity, alkaline phosphatase activity,triphenyltetrazolium chloride (TTC) reduction, and pyrrolidonyl,L-arginine, and L-aspartate arylamidase activity.

Tolerance to 1% glycine (Merck, Darmstadt, Germany) and 1.5% NaCl(Merck) was tested on tryptic blood agar base (Oxoid) supplemented with10% horse blood, as recommended by the Cape Town protocol forCampylobacteriaceae and Helicobacters. Tolerance to ox bile was testedby plating the bacteria on unsalted MacConkey agar (Oxoid).

Susceptibility to metronidazole, ampicilline, clarithromycin,tetracyclin, enrofloxacin, lincomycin, tylosin, neomycin, spectinomycinand gentamicin was tested through the agar dilution method, usingMueller-Hinton agar (Oxoid) supplemented with 10% horse blood, aspreviously described (Van den Bulck et al. (2005b) Antimicrob AgentsChemother 49, 2997-3000). All antibiotics were supplied by Sigma (St.Louis, Mo., USA) as standard powders with known potencies, except forenrofloxacin, purchased from Bayer (Brussels, Belgium). All growth andtolerance test preparations were incubated for 7 days in a micro-aerobicatmosphere at 37° C.

Growth of the organism was tested on BHI blood agar, Brucella blood agar(Oxoid) and Mueller-Hinton blood agar. Growth at 25, 30, 37, and 42° C.was determined on BHI blood agar. These media were incubated for 7 daysin a micro-aerobic atmosphere at 37° C. In addition, growth onblood-supplemented BHI agar was tested in an aerobic, aerobic with 5%CO₂, micro-aerobic and an anaerobic environment.

For polyacrylamide gel electrophoresis (PAGE) of whole-cell proteins,strain JKM4^(T) (Helicobacter cynogastricus) was grown on Mueller-Hintonagar (Oxoid) supplemented with 5% (vol/vol) horse blood and wasincubated at 37° C. in a microaerobic atmosphere containingapproximately 5% O₂, 3.5% CO₂, 7.5% H₂, and 84% N₂. A whole-cell proteinextract was prepared and sodium dodecyl sulphate PAGE was performed asdescribed previously (Pot, et al. (1994) J Appl Bacteriol 77, 362-369.).Whole-cell protein profiles of H. bizzozeronii, H. salomonis and H.felis reference strains and of type and reference strains of otherHelicobacter species were available from previous studies. Thedensitometric analysis, normalization and interpolation of the proteinprofiles, and numerical analysis were performed using the GelComparsoftware package version 4.2 (Applied Maths, Sint Martens Latem,Belgium). The similarity between all pairs of traces was expressed bythe Pearson product moment correlation coefficient presented aspercentages of similarity.

Genotypic Analysis

Genomic DNA was extracted using the DNeasy Tissue kit (Qiagen, Venlo,The Netherlands) according to the instructions of the manufacturer.

The 16S rRNA gene was amplified using primers complementary to theconserved edges. Consensus primers alpha-beta-NOT (5′-TCA MC TAG GAC CGAGTC) [SEQ ID NO:3] and omega-MB (5′-TAC CTT GTT ACT TCA CCC CA) [SEQ IDNO:4] were used, as previously described (Baele et al. (2001) J ApplMicrobiol 91, 488-491). A 1500 bp amplicon [SEQ ID NO: 5] encoding apart of 16S rRNA (FIG. 11) was amplified and sequenced using primer pD(5′-GTA TTA CCG CGG CTG CTG-3′) [SEQ ID NO:6], primer gamma* (5′-CTC CTACGG GAG GCA GCA GT-3′) [SEQ ID NO:7], primer 3 (5′-GTT GCG CTC GTT GCGGGA CT-3′) [SEQ ID NO:8] and primer O* (5′-MC TCA MG GM TTG ACG G-3′)[SEQ ID NO:9], as described elsewhere (Coenye et al. (1999) Int J SystEvol Microbiol 49, 405-413). Sequence analysis was performed using theABI Prism™ 3100 Genetic Analyzer (Applied Biosystems, Lennik, Belgium)and sequences were compared with Genbank using the BLAST algorithm.Phylogenetic analysis was performed using KODON (Applied Maths,Sint-Martens-Latem, Belgium). Pairwise alignment homologies werecalculated and a dendrogram was constructed using the neighbour-joiningmethod.

For the detection of the urease gene, a PCR with primers U430f(5′-ckgawttgatgcaagaagg-3′) [SEQ ID NO:10] and U1735r(5′-cttcgtgrattttaarrccaat-3′) [SEQ ID NO:11]was performed. This PCRresults in an amplicon of 1224 bp in H. felis, H. bizzozeronii, H.salomonis and “Candidatus H. heilmannii” (O'Rourke et al. (2004) Int JSyst Evol Microbiol 54, 2203-2211). A PCR with primers Hh2f and Hh2rwhich specifically amplifies a part of the urease gene of “Candidatus H.heilmannii” was also applied (O'Rourke et al. (2004) cited above). DNAfrom “Candidatus H. heilmannii” served as positive control, while highlypurified water was included as negative control. PCR products wereseperated through gel electrophoresis as previously described (Baele etal. (2004) J Clin Microbiol 42, 1115-1122.). Additionally, obtained PCRproducts for “Candidatus H. heilmannii” and JKM4T (Helicobactercynogastricus) were sequenced using the BigDye Terminator CycleSequencing Kit (Perkin Elmer, Applied Biosystems) on a ABI Prism™ 3100Genetic Analyzer (Applied Biosystems). The electropherograms wereexported and converted to Kodon (Applied Maths) and sequences werecompared with Genbank using BLAST.

tRNA intergenic length polymorphism analysis (tDNA-PCR) was performedwith a consensus primer T3B (5′-AGG TCG CGG GTT CGA ATC C-3′) [SEQID:12] (labeled with the fluorescent marker TET) and primer HT135R(5′-ACC AAC TGG GCT AAG CGA CC-3′) [SEQ ID NO:13], a specific primercomplementary to the tRNA intergenic spacer of Helicobacter species, asdescribed earlier (Baele et al., 2004 cited above). DNA extracted frompure cultures of H. felis, H. salomonis and H. bizzozeronii served aspositive controls, while highly purified water was included as negativecontrol. The PCR products were separated by means of capillaryelectrophoresis using the ABI Prism™ 3100 Genetic Analyzer (AppliedBiosystems, Lennik, Belgium). Lengths were determined by interpolationwith an internal size standard mixture of GeneScan 500 ROX (AppliedBiosystems) and GeneScan 400-HD ROX (Applied Biosystems), usingGeneMapper (Applied Biosystems).

To determine the prevalence of the new Helicobacter species in cats anddogs, gastric samples were collected from the corpus region of 110 dogs(65 male, 45 female, age ranging from 1 day to 18 years) and 43 cats (25male, 18 female, aged from 7 weeks to 18 years), from various breeds,which were presented for autopsy at the Department of Pathology (Facultyof Veterinary Medicine, Ghent University) between November 2001 andSeptember 2003 with various pathology. DNA was extracted from the felineand canine samples using the DNeasy tissue kit (Qiagen), according tothe instructions of the manufacturer.

These DNA samples were subjected to tRNA intergenic length polymorphismanalysis.

Phenotypic studies The salient tests that distinguish the new isolatefrom other canine gastric helicobacters are listed in Table 4. TABLE 4Characteristics of JKM4 (Helicobacter cynogastricus) and related gastricHelicobacters Characteristic H. cynogastricus H. felis H. bizzozeroniiH. salomonis H. pylori Cell length (μm) 10-18   5-7.5  5-10 5-7 2.5-5  Cell width (μm) 0.8-1.0 0.4 0.3  0.8-0.12 0.5-1.0 Periplasmicfibrils + + − − − Location of the flagella bipolar bipolar bipolarbipolar polar No. of flagella  6-12 14-20 10-20 10-23 4-8 Flagellarsheath + + + + + Catalase activity + + + + + Oxidase activity + + + + +Urease activity + + + + + Nitrate reduction + + + + − Hippuratehydrolysis − − − − − Indoxyl acetate hydrolysis − − + + − γ-Glutamylaminopeptidase + + + + + TTC reduction + − + + + Alkaline phosphataseactivity + + + + + Growth at 25° C. − − − − − 37° C. + + + + + 42° C. −− + − − Tolerance to 1% ox bile − − − − − 1.5% NaCl − − − − − 1% glycine− − − − −

The ultrastructural studies of strain JKM4^(T) (Helicobactercynogastricus) revealed large spiral cells which were 10 to 18 μm longand approximately 1 μm wide, with three to eight spirals per cell. Oneperiplasmic fibril was present at every bacterial cell, running alongthe external side of the helix. Up to 12 sheathed flagella were detectedat both ends of each cell and these flagella were slightly off-centre.The flagellae were blunt-ended and the terminal diameter was wider thanthe average diameter of the flagellar body. Coccoid forms were observedin older cultures. The ultrastructural characteristics of the organismswere examined several times after several subcultures and were the samein all studies.

The isolate presented oxidase, urease and catalase activity, and did nothydrolyse indoxyl acetate. The organisms were able to reduce nitrate andTTC, and were positive in the esterase, gamma-glutamyl transpeptidase,L-arginine arylamidase, and alkaline phosphatase tests, but negative inthe hippurate hydrolysis, pyrrolidonyl arylamidase, and L-aspartatearylamidase tests. The bacteria grew well on blood-supplemented BHI,brucella and Mueller-Hinton agar media.

The bacteria were sensitive to all antimicrobials tested, as indicatedby low MIC values, ranging from 0.03 to 0.25 μg/mi. They did not grow onmedia containing 1.5% NaCl, 1% bile or 1% glycine. They were able togrow at 37° C. and 30° C., but not at 25° and 42° C. Growth was abled inboth anaerobic and microaerobic environments, while atmospherescontaining normal levels of oxygen or solely an increase of CO₂ were notsuitable to culture the bacteria.

The whole-cell protein profile of strain JKM4^(T) (Helicobactercynogastricus) differed considerably from those of reference strains ofother Helicobacter species (FIG. 9A). Correlation levels towards theprotein profiles of other Helicobacter reference strains were all below0.80 indicating that strain JKM4^(T) represents a novel Helicobacterspecies. FIG. 9B shows the result of the numerical analysis of theprotein profiles of strain JKM4^(T) (Helicobacter cynogastricus) and itsnearest phylogenetic neighbours.

Genotypic Studies

Sequencing of the 16S rRNA gene of JKM4^(T) (Helicobacter cynogastricus)revealed >97% homology with H. felis, H. bizzozeronii, H. salomonis and“Candidatus H. heilmannii”, while the sequence differed more than 3% of“Candidatus H. suis” (FIG. 9B). A phylogenetic tree revealed clusteringof the new isolate within all these species (FIG. 10).

PCR on genomic DNA of JKM4^(T) (Helicobacter cynogastricus) usingprimers U430f (5′-gckgawttgatgcaagaagg-3′) [SEQ ID NO:14 and U1735r(5′-cttcgtgrattttaarrccaat-3′) [SEQ ID NO:15] produced a series ofaspecific fragments and not the expected fragment of 1224 bp. PCR with“Candidatus H. heilmannii” specific primers Hh2f and Hh2r resulted inthe production of a 320 bp fragment, which consistently differed fromthe expected 380 bp produced from DNA of “Candidatus H. heilmannii”.Sequence analysis of the PCR product revealed a unique sequence, whichdid not match to any sequence in the Genbank.

Analysis of the PCR products produced from the DNA of the new isolate inthe tDNA-PCR consistently revealed an amplicon of 136.6 bp, whichdiffered from the tDNA-amplicon of H. felis (137 bp), H. bizzozeronii(136 bp) and H. salomonis (134 bp). A fragment of the same size wasfound in 1 cat (2.3%) and in 23 dogs (20.9%).

The present example demonstrates the existence of a fourth culturableHelicobacter species, able to colonise the canine stomach.

Analysis of the 16S rRNA gene revealed a high degree of homology withthe three previously isolated carnivorous Helicobacter species.

The urease gene has recently been approved to be discriminative betweenthese Helicobacter species. PCR on genomic DNA of isolate JKM4^(T)(Helicobacter cynogastricus) using primers that detect the urease genein H. felis, H. bizzozeronii and H. salomonis only produced aspecificfragments. A “Candidatus H. heilmannii” specific PCR did revealed a PCRfragment but with a sequence different to the one of Candidatus H.heilmannii”.

In addition, the PCR amplicon of the novel species of the presentinvention by tDNA-PCR differs from the amplicons of other Helicobacters.These findings, together with the results of protein-profiling, whichrevealed a completely different pattern from the patterns of otherHelicobacter species, demonstrate that isolate JKM4^(T) is a distinct,novel Helicobacter species which we designate Helicobactercynogastricus.

1. A method of vaccinating an animal against a Candidatus Helicobactersuis infection comprising administering to said animal a vaccinecomprising one or more antigen preparations from one or more bacterialspecies, wherein said bacterial species are related to CandidatusHelicobacter suis.
 2. The method according to claim 1, wherein saidbacterial species related to Candidatus Helicobacter suis is/are speciesof bacteria having a 16S rRNA sequence having at least 93% sequenceidentity to the sequence of Candidatus Helicobacter suis.
 3. The methodaccording to claim 1, wherein said species related to CandidatusHelicobacter suis is/are selected from the group consisting ofHelicobacter felis, Helicobacter salomonis, Helicobacter heilmannii(type II), Helicobacter cynogastricus, Helicobacter pylori orHelicobacter bizzozeronii.
 4. The method according to claim 1, whereinsaid species related to Candidatus Helicobacter suis is Helicobacterfelis or Helicobacter bizzozeronii.
 5. The method according to claim 1,wherein said antigen preparation comprises whole-killed bacteria.
 6. Themethod according to claim 1, wherein said antigen preparation compriseslive-attenuated bacteria.
 7. The method according to claim 1, whereinsaid antigen preparation is comprises a processed and/or artificialbacterial preparation.
 8. The method according to claim 1, wherein saidantigen preparation comprises a bacterial lysate.
 9. The methodaccording to claim 1, wherein said vaccine further comprises anadjuvant.
 10. The method according to claim 1, wherein said vaccine isadministered intranasally.
 11. The method according to claim 1, whereinsaid vaccine is administered subcutaneously or intramuscularly.
 12. Themethod according to claim 1, wherein said vaccine is administeredprophylactically.
 13. The method according to claim 1, wherein saidvaccine is administered therapeutically.
 14. A vaccine for immunizationagainst Candidatus Helicobacter suis comprising a composition of one ormore antigen preparations from one or more bacterial species, whereinsaid bacterial species is a species related to Candidatus Helicobactersuis.
 15. The vaccine according to claim 14, wherein said bacterialspecies related to Candidatus Helicobacter suis is/are species ofbacteria having a 16S rRNA sequence with at least 93% sequence identityto the sequence of Candidatus Helicobacter suis.
 16. The vaccineaccording to claim 14, wherein said species related to CandidatusHelicobacter suis is/are selected from the group consisting ofHelicobacter felis, Helicobacter salomonis, Helicobacter heilmannii(type II), Helicobacter cynogastricus, Helicobacter pylori orHelicobacter bizzozeronii.
 17. The vaccine according to claim 14,wherein said species related to Candidatus Helicobacter suis isHelicobacter felis or Helicobacter bizzozeronii.
 18. The vaccineaccording to claim 14, wherein said species related to CandidatusHelicobacter suis is Helicobacter cynogastricus.
 19. The vaccineaccording to claim 14, wherein said antigen preparation compriseswhole-killed bacteria.
 20. The vaccine according to claim 14, whereinsaid antigen preparation comprises live-attenuated bacteria.
 21. Thevaccine according to claim 14, wherein said antigen preparationcomprises a processed and/or artificial bacterial preparation.
 22. Thevaccine according to claim 14, wherein said antigen preparationcomprises a bacterial lysate.
 23. The vaccine according to claim 14,wherein said vaccine further comprises an adjuvant.
 24. A method forobtaining an animal model for Candidatus Helicobacter suis infection ina laboratory or model animal comprising: a) isolating cells from thestomach wall of a pig infected with Candidatus Helicobacter suis, b)making a homogenate of said cells, and c) infecting said laboratory ormodel animal intragastrically with said homogenate.
 25. An isolatedbacterium of the species Helicobacter cynogastricus deposited underAccession Number LMG P-23100.
 26. A method for the production of avaccine comprising the step of preparing an antigen preparation of anisolate of the species Helicobacter cynogastricus.
 27. An in vitromethod for the detection of a Candidatus H. suis infection in an animalcomprising the steps of: a) providing a faecal sample of said animal, b)isolating DNA from said faecal sample, c) amplifying a fragment of the16S rRNA gene of Candidatus H. suis, d) detecting the presence of anamplified fragment.
 28. The method of claim 27, wherein said fragment isa fragment of less than 400 bp.
 29. The method according to claim 27,wherein the amplification is performed with primers having SEQ ID NO:1and SEQ ID NO:2.